Worm Breeder's Gazette 11(5): 39

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Progress on the Molecular Characterization of the unc-44 Gene

Anthony Otsuka, Bin Yang, Lan Tang and Kyu-Hwang Shim

Figure 1

Mutations in the unc-44 gene affect the direction of axonal 
outgrowth and result in fasciculation of nerves with inappropriate 
partners [(Hedgecock et al., Dev.  Biol., 111, 158-179 (1985)].  We 
have cloned the unc-44 gene by transposon tagging and have partially 
characterized the nature of six transposon insertion mutations.  
Results of Southern Blot Analysis of unc-44 Mutations.  The putative 
unc-44 insertion mutations appear to result in changes in two 
different regions (Sites A and B) separated by approximately 10 kb.  
The cDNA clones DD#PA049 and DD#PA066 are found at Site A and DD#PA013 
at Site B.
The unc-44(rh1013) allele is an insertion at Site A.  The unc-44(
rh1013) mutation is due to a 1.6 kb insertion.  The presence of a DNA 
insertion, rather than an alteration of a restriction site, is 
indicated by restriction fragment size differences with either EcoRI 
or BamHI digests probed with cosmid clone B0350 or cDNA clone A066.  
The presence of a SalI site in the inserted DNA suggests that it is a 
Tc1 element.  The rh1013 insertion is included in the region covered 
by the cDNA clone, A066.  Southern blot analysis of two independent 
revertants reveals a wild type pattern with B0350 and A066 probes.  
The unc-44(q331) allele is an insertion at Site A.  The unc-44(q331) 
mutation is due to a 1.6 kb insertion at a site distinct from rh1013.  
Again, digestion with either EcoRI or BamHI reveals a 1.6 kb insertion 
containing a SalI site and is consistent with a Tc1 insertion.  The 
region of the q331 insertion is not included in the A066 cDNA clone.  
A single revertant (q331, q332) yields a wild type pattern.  
The unc-44(rh1042) mutation is associated with an insertion at Site 
B.  The rh1042 allele is associated with a Tc1 insertion (determined 
by DNA sequencing) in an open reading frame.  The open reading frame 
is included in the cDNA clone A013.  The Tc1 element is not excised in 
the single revertant analyzed, but changes occur in a nearby 
restriction fragment.  
The unc-44(st200) and mn259 alleles cause changes at Site B.  The 
st200 and mn259 alleles result in changes in two BamHI fragments at 
Site B.  The size changes are consistent with one insertion of 3.2 kb 
or two insertions of 1.6 kb each, but the exact nature of the 
mutations and reversions remains to be determined.  
The unc-44(mn339) allele results from a 2.1 kb insertion at Site B.  
The mn339 allele results in the loss of a 2.1 kb BamHI fragment and 
the acquisition of a 4.2 kb fragment.  The single revertant analyzed 
results from a 4.3 kb deletion (the 4.2 and 0.75 kb BamHI fragments in 
the mutant are replaced by a 0.8 kb fragment in the revertant).  
Sequence analysis of unc-44 cDNAs.  DNA sequence analysis A049 and 
A066 clones reveals nearly identical sequences in the central region 
with divergence at the 5' and 3' ends.  A013 does not appear to be 
related in sequence to A049 or A066.
[See Figure 1]

Figure 1